Kv1.1 preserves the neural stem cell pool and facilitates neuron maturation during adult hippocampal neurogenesis

Significance Despite decades of research on adult neurogenesis, little is known about the role of bioelectric signaling in this process. In this study, we describe how a voltage-gated potassium channel, Kv1.1, supports adult neurogenesis by maintaining the neural stem cell niche and facilitating newborn neuron development. Additionally, we show that deletion of Kv1.1 from adult neural stem cells contributes to modest impairments in hippocampus-dependent contextual fear learning and memory. Dysfunctional adult neurogenesis has been implicated in cognitive decline associated with aging and neurological disease. Therefore, understanding the role of Kv1.1 in adult neurogenesis represents an opportunity to identify new therapeutic targets to promote healthy neurogenesis and cognition.

In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel K v 1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of K v 1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove K v 1.1 from adult neural stem cells via tamoxifen administration. We determined that K v 1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.
voltage-gated potassium channel j adult neurogenesis j hippocampus j learning and memory The subgranular zone of the hippocampus is one of two well-characterized neurogenic niches in the adult mouse brain. Integration of adult-born granule cells into the dentate gyrus is important for learning and memory, and impaired adult neurogenesis has been implicated in neurodegenerative and neuropsychiatric diseases (1)(2)(3)(4)(5)(6). Adult hippocampal neurogenesis is divided into several developmental stages. Initially, quiescent neural stem cells with radial glia-like morphology-known as radial glia-like neural stem cells (type 1 cells)-activate and proliferate, either self-renewing or differentiating into intermediate neural progenitor cells with a glia-like phenotype (type 2a cells). As these cells differentiate, they lose their stem cell properties and display more neuron-like features (type 2b cells). Then, they develop into neuroblasts (type 3 cells) within a few days. Over the course of the next 2 wk to 4 wk, type 3 cells give rise to immature adult-born neurons that extend an apical dendrite into the dentate granule cell layer and grow secondary and tertiary dendrites. Simultaneously, they migrate from the subgranular zone into the dentate granule cell layer. Finally, adult-born granule cells mature into highly excitable neurons and integrate into the dentate gyrus circuitry (1)(2)(3)(4)(5)(6)(7)(8).
While adult hippocampal neurogenesis is regulated by various environmental and endogenous factors, recent studies have also begun to explore the role of bioelectric signaling in this process. In nonexcitable cells, such as neural stem and progenitor cells, changes in the membrane potential can orchestrate proliferation, differentiation, migration, and survival during development (9). The membrane potential is controlled by ion channels, and ion channel dysfunction often results in neurodevelopmental disorders (10,11). Interestingly, ion channels continue to modulate the membrane potential and cell dynamics of neural stem and progenitor cells during postnatal neurogenesis (12)(13)(14)(15)(16)(17)(18)(19). In adult radial glia-like neural stem cells, gap junctions and inward rectifying potassium channels maintain the membrane potential (14,17,19). Their proliferation is also regulated by GABAergic and glutamatergic signaling, where these neurotransmitters activate their corresponding ligand-gated ion channels to alter the membrane potential (13,15,18). Additionally, local circuit activity is critical for young adultborn granule cells, which receive, in order, depolarizing GABAergic inputs, excitatory glutamatergic inputs, and, finally, inhibitory GABAergic inputs to advance through stages of maturation and survival (20)(21)(22)(23).
In this study, we examine the role of the voltage-gated potassium channel K v 1.1 in adult hippocampal neurogenesis. K v 1.1 is encoded by the Kcna1 gene in mice, and its expression begins increasing at ∼2 wk after birth and stabilizes in adulthood (24). K v 1.1 is well known for its role in regulating neuronal excitability and seizure activity (25,26). Mice without functional K v 1.1-K v 1.1 null mutant mice and megencephaly Significance Despite decades of research on adult neurogenesis, little is known about the role of bioelectric signaling in this process. In this study, we describe how a voltagegated potassium channel, K v 1.1, supports adult neurogenesis by maintaining the neural stem cell niche and facilitating newborn neuron development. Additionally, we show that deletion of K v 1.1 from adult neural stem cells contributes to modest impairments in hippocampusdependent contextual fear learning and memory. Dysfunctional adult neurogenesis has been implicated in cognitive decline associated with aging and neurological disease. Therefore, understanding the role of K v 1.1 in adult neurogenesis represents an opportunity to identify new therapeutic targets to promote healthy neurogenesis and cognition.
To address this issue and clarify the role of K v 1.1 in adult hippocampal neurogenesis, we created inducible K v 1.1 conditional knockout (K v 1.1 cKO) mice, which allowed us to specifically delete Kcna1 from adult neural stem cells via tamoxifen injection and eliminate the confounding effect of seizures in our study. Using this mouse model, we first removed K v 1.1 in neonatal neural stem cells to validate our previous results with improved temporal resolution. Indeed, we recapitulated our previous observations showing that loss of K v 1.1 in neonatal neural stem cells increases proliferation and neuron production. Interestingly, the role of K v 1.1 in adult neural stem cells is more complex. We discovered that K v 1.1 prevents overproliferation and depletion of radial glia-like neural stem cells and enables proper adult-born granule cell maturation and positioning during adult neurogenesis. We further corroborated our findings of an age-dependent role of K v 1.1 using MADM of heterozygous K v 1.1 (Kcna1 +/À ) mice (33)(34)(35). Finally, we determined that decreased adult neurogenesis in K v 1.1 cKO mice causes deficits in hippocampus-dependent contextual fear conditioning. These results demonstrate that K v 1.1 expression in adult neural stem cells is integral for preserving hippocampal neurogenesis and contextual learning and memory.

Results
Time-Controlled Deletion of K v 1.1 from Neural Stem Cells. To investigate the function of K v 1.1 in neural stem cells at various postnatal stages, we generated K v 1.1 cKO mice. We bred mice expressing a tamoxifen-inducible Cre recombinase (Cre) in neural stem cells (Nestin-Cre ERT2 ) (38)(39)(40) with Kcna1 floxed mice (Kcna1 fl/fl ) (41) and Cre reporter mice (PC::G5-tdT) (42) to achieve temporal and cell type-specific control of Kcna1 deletion. Upon tamoxifen injection, Cre begins expressing in neural stem cells, resulting in the removal of K v 1.1 and expression of tdTomato and GCaMP5G in the neural stem cells and their progeny. While the Cre-expressing neural stem cells are a small subset of all cell types in the dentate gyrus, the expression of tdTomato and GCaMP5G in these cells enables us to identify them for lineage tracing. We amplified the GCaMP5G signal with an anti-GFP antibody, because the anti-GFP antibody was compatible with our histology techniques using multiple cell markers. In this way, we successfully read out Cre expression in neural stem cells. To control for the possible effects of tamoxifen, Cre, and reporter expression on neural stem cell dynamics, we bred mice with wild-type Kcna1 (Kcna1 +/+ ) with Nestin-Cre ERT2 and PC::G5-tdT mice for our control cohort (K v 1.1 WT) (SI Appendix, Fig. S1A).
To validate K v 1.1 knock-out after tamoxifen injection, we injected 8-wk-old K v 1.1 cKO and K v 1.1 WT mice with tamoxifen for three consecutive days and used fluorescence-activated cell sorting to isolate Cre-expressing tdTomato+ cells from the dentate gyrus at 2 wk post tamoxifen injection (SI Appendix, Fig. S1 B-E). We found that Kcna1 messenger RNA expression was decreased by ∼90% in tdTomato+ cells in the dentate gyrus of K v 1.1 cKO mice compared to those of K v 1.1 WT mice (P = 0.0001) (SI Appendix, Fig. S1F). We also recorded the resting membrane potential of acutely dissociated tdTomato+ cells from the dentate gyrus at 2 wk post tamoxifen injection to determine whether K v 1.1 has been functionally deleted (SI Appendix, Fig. S2A). To assess the effect of acute K v 1.1 inhibition on resting membrane potential, we applied the selective K v 1.1 blocker, Dendrotoxin-K (DTx-K), to tdTomato+ K v 1.1 WT cells and observed a depolarized resting membrane potential (À70 ± 2.6 mV) compared to untreated K v 1.1 WT cells (À87 ± 0.58 mV) (P = 0.0002) (SI Appendix, Fig. S2 B and C). Consistent with our findings using DTx-K for acute K v 1.1 inhibition, we found that the resting membrane potential of tdTomato+ K v 1.1 cKO cells was similarly depolarized (À68 ± 1.0 mV) compared to K v 1.1 WT cells (P = 0.0002). Together, these results show that K v 1.1 is functionally knocked out of the Cre-expressing tdTomato+ neural stem cell lineage of K v 1.1 cKO mice by 2 wk post tamoxifen injection, likely resulting in depolarized cells.

Conditional Knockout of K v 1.1 in Neonatal Neural Stem Cells
Increases Early Postnatal Hippocampal Neurogenesis. Both K v 1.1 null mice and mceph mutant mice display increased neonatal neurogenesis before seizure onset around 1 mo after birth (12,(27)(28)(29)(30)(31)(32). As K v 1.1 cKO mice allowed us to examine the role of K v 1.1 in early postnatal neurogenesis with more precise temporal resolution, we focused on the role of K v 1.1 during peak hippocampal development at postnatal day 7 (P7), just before the second postnatal week when hippocampal neurogenesis begins transitioning from a more embryonic stage to adult stage (43,44).
We injected tamoxifen at P0 to knock out K v 1.1 and conducted lineage tracing by injecting Bromodeoxyuridine (BrdU), which is incorporated into the DNA of actively dividing cells (45)(46)(47)(48), at P7. We then quantified the number of progeny cells in the dentate gyrus of K v 1.1 cKO mice and K v 1.1 WT mice at P14 (SI Appendix, Fig. S3A). To determine which cell types were altered in the Cre-expressing GFP+ neural stem cell lineage, we costained the sections with established neural stem and progenitor cell marker, Sox2, and the postmitotic neuronal marker, NeuN. We found that neural stem and progenitor cell progenies from cells dividing at P7 (GFP+, BrdU+, Sox2+) in K v 1.1 cKO subgranular zone were increased by ∼60% (P = 0.036) (SI Appendix, Fig. S3 B and C). Within the dentate granule cell layer, neurons produced from cells dividing at P7 (GFP+, BrdU+, NeuN+) were increased by ∼55% (P = 0.0007) (SI Appendix, Fig. S3 D and E). The enhanced neonatal neurogenesis that we observed in K v 1.1 cKO mice is similar to our previous findings in K v 1.1 null mice (12), providing further evidence that K v 1.1 acts as a brake on early postnatal neurogenesis.
Deletion of K v 1.1 in Adult Neural Stem Cells Leads to a Transient Activation Followed by a Depletion of Radial Glia-Like Cells. Next, we investigated the role of K v 1.1 in adult hippocampal neurogenesis. To specifically ablate K v 1.1 in the adult neural stem cell lineage, we injected 8-wk-old adult mice with tamoxifen for three consecutive days. Unlike the K v 1.1 null mice and mceph mutant mice, adult K v 1.1 cKO mice injected with tamoxifen did not display seizure phenotypes, thereby allowing us to eliminate the confounding effects of seizures on adult neurogenesis from our study.
We started by examining mice 4 wk after tamoxifen injection to assess the early effects of K v 1.1 deletion on adult quiescent radial glia-like neural stem cells (type 1 cells), which are labeled by Sox2 and the glial marker, GFAP. As they become activated, radial glia-like neural stem cells begin expressing the mitotic marker, MCM2 (Fig. 1A) (49). At 4 wk post tamoxifen injection, quiescent (GFP+, GFAP+, Sox2+, MCM2À) and activated (GFP+, GFAP+, Sox2+, MCM2+) radial glia-like neural stem cells were increased by ∼100% (P = 0.048) and ∼80% (P = 0.046), respectively, in K v 1.1 cKO mice as compared to K v 1.1 WT mice ( Fig. 1 B-E). This suggests that loss of K v 1.1 initially promotes both radial glia-like neural stem cell division and self-renewal. Instead of self-renewing, radial glialike neural stem cells can also divide and differentiate into type 2a cells, losing their GFAP expression (SI Appendix, Fig. S4A).
To determine whether loss of K v 1.1 alters radial glia-like neural stem cell differentiation, we quantified the amount of type 2a cells (GFP+, GFAPÀ, Sox2+, MCM2+) and found a trend toward statistical significance that type 2a cells in K v 1.1 cKO mice were increased by ∼70% at 4 wk post tamoxifen injection (P = 0.068) (SI Appendix, Fig. S4 B and D). It is possible that the trend toward an increase of type 2a cells arose from either enhanced radial glia-like neural stem cell proliferation, which pushed radial glia-like neural stem cells to both self-renew and differentiate, or increased proliferation of both radial glia-like neural stem cells and type 2a cells in K v 1.1 cKO mice. Interestingly, the increase in radial glia-like neural stem cells and type 2a cells did not lead to additional type 2b and proliferating type 3 cells (GFP+, GFAPÀ, Sox2À, MCM2+) (SI Appendix, Fig. S4 C and D). From these observations, K v 1.1 expression seems to discourage adult radial glia-like neural stem cell division.
To determine the long-term effects of K v 1.1 deletion, we investigated changes in the neural stem cell lineage at 8 wk after tamoxifen injection. Surprisingly, quiescent (GFP+, GFAP+, Sox2+, MCM2À) and activated (GFP+, GFAP+, Sox2+, MCM2+) radial glia-like neural stem cells were reduced by ∼40% (P = 0.039) and ∼65% (P = 0.016), respectively, in K v 1.1 cKO mice as compared to K v 1.1 WT mice ( Fig. 1 F-H). We interpret this to mean that the initial increase in radial glialike neural stem cell proliferation eventually exhausted their ability to self-renew and depleted the radial glia-like neural stem cell pool. We also examined the role of K v 1.1 in type 2a and proliferating type 2b/3 cells. Because of the variability of the type 2a (GFP+, GFAPÀ, Sox2+, MCM2+) cell counts, we were unable to determine, with confidence, whether they were altered in the K v 1.1 cKO mice at 8 wk post tamoxifen injection (P = 0.14) (SI Appendix, Fig. S4 E and G). We did not find a difference in type 2b/3 cells (GFP+, GFAPÀ, Sox2À, MCM2+) between K v 1.1 cKO mice and K v 1.1 WT mice at 8 wk post tamoxifen injection (SI Appendix, Fig. S4 F and G). Taken together, these results indicate that K v 1.1 acts as a brake on overproliferation to prevent early depletion of the neurogenic stem cell pool. To investigate the role of K v 1.1 in later stages of adult-born granule cell production, we stained for a neurogenesis marker, doublecortin (DCX) at 8 wk post tamoxifen injection. DCX begins to express in a subset of type 2b cells and ceases to express as they become NeuN+ mature neurons (50)(51)(52). Interestingly, there was a ∼55% decrease in GFP+, DCX+ cells (P = 0.041) (Fig. 2 A and D) in K v 1.1 cKO mice although the amount of proliferating type 2b/3 cells was not altered (SI Appendix, Fig. S4F). This raises the question whether the observed decrease in GFP+, DCX+ cells was due to the altered development of young adult-born granule cells. We relied on the distinct morphology of DCX+ cells at different stages of maturation to identify more-developed DCX+ adult-born granule cells as those with tertiary dendrites (50)(51)(52). Interestingly, in the K v 1.1 cKO lineage, there was a ∼75% decrease in the number of GFP+, DCX+ cells with tertiary dendrites (P = 0.012) as well as a ∼45% decrease in the proportion of more-developed GFP+, DCX+ cells with tertiary dendrites among all GFP+, DCX+ cells (P = 0.017) (Fig. 2 B-D). Together, these results indicate that loss of K v 1.1 hinders young adult-born granule cell maturation.
Those K v 1.1 cKO neurons that successfully matured were also more likely to be inappropriately positioned. As young adult-born granule cells mature, they migrate from the subgranular zone into the dentate granule cell layer such that a majority are positioned within the inner two-thirds of the dentate granule cell layer (Fig. 2E) (20,53). The percentage of mature GFP+ adult-born granule cells (GFP+, NeuN+) found in the outer third of the dentate granule cell layer at 8 wk post tamoxifen injection in K v 1.1 cKO mice was ∼215% higher than that of K v 1.1 WT mice (P = 0.033) (Fig. 2 F and H), indicating that loss of K v 1.1 impairs adult-born granule cell positioning. These findings point toward a critical role of K v 1.1 in facilitating successful adult-born granule cell development, as aberrant migration and positioning of adult-born granule cells has been found in mouse models of traumatic brain injury, schizophrenia, and neurodegeneration (54)(55)(56). These findings may also explain why we observed a ∼50% reduction in mature GFP+ adult-born granule cells (GFP+, NeuN+) in K v 1.1 cKO mice by 8 wk post tamoxifen injection (P = 0.020) (Fig. 2 G and H ). As young adult-born granule cells from neural stem cells lacking K v 1.1 cannot properly mature and position themselves, they are likely unable to successfully integrate into the dentate gyrus circuitry. Taken together, our observations indicate that K v 1.1 is integral for adult-born granule cells to develop proper morphology and positioning, which would allow them to incorporate synaptic inputs, integrate into the hippocampal circuitry, and fulfill their critical functions in learning and memory.

MADM Analyses Reveal a Transient Increase of Neural Stem
Cell Lineage Lacking K v 1.1. In our previous studies, we performed MADM (33-35) with heterozygous K v 1.1 (Kcna1 +/À ) mice, in which sparse somatic recombination driven by constitutively active Nestin-Cre generates a subpopulation of neural stem cells that lack K v 1.1 (Nestin-Cre;Kcna1 +/À ;MADM-6) ( Fig. 3A) (12). Homozygous K v 1.1 null neural stem cell lineages are marked with GFP, and homozygous K v 1.1 wild-type neural stem cell lineages are marked with tdTomato. Using this model, we observed ∼180% increase in progeny neurons from K v 1.1 null neural stem cells in the dentate granule cell layer of 2to 3-mo-old Nestin-Cre;Kcna1 +/À ;MADM-6 mice (P < 0.0001) [ Fig. 3 B and C; data from 1-mo-old and 2-to 3-mo-old cohorts originally published in figure 1 of Chou et al. (12)]. This is consistent with our findings from K v 1.1 null mice and K v 1.1 cKO mice that loss of K v 1.1 in neonatal neural stem cells promotes neonatal radial glia-like neural stem cell proliferation and neuronal production (SI Appendix, Fig. S3) (12). In 2-to 3-mo-old Nestin-Cre;Kcna1 +/À ;MADM-6 mice, presumably, a large population of neonatal-born neurons remains, as neonatal-born neurons begin apoptosis ∼2 mo after birth (57)(58)(59). However, once the mice have reached 6 mo to 13 mo of age, neonatal-born K v 1.1 null progeny neurons would have been trimmed via apoptosis and therefore appear as a smaller portion of the GFP+ population. Indeed, we found no increase in K v 1.1 null progeny neurons in the dentate granule cell layer of 6-to 13-mo-old mice (Fig. 3 B and C). As the loss of K v 1.1 negatively impacts adult neurogenesis (Fig. 2) The hippocampus is important for contextual learning and memory; decreased hippocampal neurogenesis has been found to impair contextual fear conditioning, where mice learn to associate an environment with fear (60-63), and pattern separation, where they learn to discriminate between two similar contexts (40, 64, 65). Since the deletion of K v 1.1 in adult neural stem cells reduced adult hippocampal neurogenesis (Fig. 2), we hypothesized that K v 1.1 cKO mice would have learning and memory deficits compared to K v 1.1 WT mice. We conducted behavioral tests on K v 1.1 cKO mice starting at ∼4 mo of age when adult neurogenesis is less variable than at 2 mo of age (66). We injected ∼4-mo-old K v 1.1 cKO mice with tamoxifen for five consecutive days to induce Cre expression and K v 1.1 deletion in more neural stem cells (Fig. 4A). In control behavioral experiments, we did not find statistically significant differences between K v 1. K v 1.1 WT mice. To test for the effects of K v 1.1 cKO on hippocampus-dependent learning and memory, we used a protocol with three segments to first examine contextual fear conditioning, then contextual recall and generalization, and, finally, contextual discrimination (pattern separation) (64,67). First, mice underwent 3 d of contextual fear conditioning where they learned to associate the fear context with a foot shock. On days 1 to 3, they were placed in the fear context, a single foot shock was administered 3 min into the session, and mice were removed 1 min after the shock. Percent freezing before (0 min to 3 min) and after (3 min to 4 min) the foot shock was measured daily (Fig. 4B). The two genotypes did not display differences in percent freezing before the foot shock on day 1, showing that they have similar freezing levels at baseline (Fig. 4C). Interestingly, we found that K v 1.1 cKO mice appeared to have reduced expression of contextual fear memory (genotype effect [F 1, 33 = 7.5, P = 0.010], day effect [F 1.8, 61 = 197, P < 0.0001], and genotype × day interaction [F 2, 66 = 2.9, P = 0.060]), which manifested as a ∼25% and ∼20% reduction in freezing time prior to foot shock as compared to K v 1.1 WT on day 2 and day 3 (post hoc analysis of linear mixed-model using Sidak's multiple comparisons test: day 2 [P = 0.10], and day 3 [P = 0.010]) (Fig. 4C). Given that there was no difference between the two genotypes in percent freezing following the shock on days 1 to 3 (Fig. 4D), this study indicates that K v 1.1 cKO mice exhibited a mild deficit in contextual fear conditioning.
Next, we demonstrated that both genotypes generalized their learned fear to a similar novel neutral context (SI Appendix, Fig. S6 A-C), thus allowing us to proceed to the third segment of our experiment and assess their ability to discriminate between the two contexts. In the pattern separation task on days 6 to 19, mice were placed in the two contexts daily, and shock was again administered in the fear context. Percent freezing was averaged for each 2-d block, to reduce variability (SI Appendix, Fig. S6D). As the mice learned to discriminate between the two contexts, they froze more in the fear context than the neutral context. We found no significant genotype effect in the pattern separation test (SI Appendix, Fig. S6 E-I).
To validate that the modest effect on contextual fear conditioning we observed was due to decreased adult hippocampal neurogenesis in K v 1.1 cKO mice, we collected brain tissues from the behavioral cohorts and stained for NeuN to assess the extent of neurogenesis in the GFP+ lineages with tamoxifen-induced Cre expression and, in K v 1.1 cKO cohort, deletion. We found that adult neurogenesis was decreased by ∼20% in K v 1.1 cKO mice as compared to K v 1.1 WT controls (P = 0.037) (Fig. 4E). These observations are consistent with previous studies reporting diminished adult neurogenesis resulting in deficient contextual fear conditioning (60)(61)(62)(63). Whereas past studies using X-ray irradiation to reduce adult neurogenesis by more than ∼90% have produced deficits in pattern separation (40,64), the ∼20% decrease in adult neurogenesis caused by the loss of K v 1.1 is probably insufficient to impair their performance in the pattern separation test. In summary, these results indicate that deletion of K v 1.1 hinders adult neurogenesis, resulting in mild impairments in contextual fear conditioning.
During early steps of adult hippocampal neurogenesis, K v 1.1 is important for preserving the radial glia-like neural stem cell (type 1 cell) pool (Fig. 5A), which is maintained by a delicate balance between radial glia-like neural stem cell quiescence and activation (71). Initially, radial glia-like neural stem cells without K v 1.1 rapidly divide to 1) self-renew, generating more quiescent radial glia-like neural stem cells (Fig. 1C), and 2) differentiate, likely producing more type 2a cells (SI Appendix, Fig. S4B). However, radial glia-like neural stem cells have varying self-renewal capacity. Over time, increased proliferation in the absence of K v 1.1 may become unsustainable and might exhaust the radial glia-like neural stem cells with limited proliferative potential as they undergo terminal differentiation and are eliminated from the progenitor pool (72,73). Interestingly, the switch between radial glia-like neural stem cell quiescence and activation can be regulated by network activity of glutamatergic mossy cells and long-range GABAergic neurons. Ablation of both cell types can produce a similar initial activation followed by depletion of radial glia-like neural stem cells (13,15,18). In addition to regulation via synaptic inputs, our study suggests that radial glia-like neural stem cells may rely on K v 1.1 channel activity to prevent their subsequent activation and depletion (SI Appendix, Fig. S2 and Fig. 1). Perhaps loss of K v 1.1 depolarizes adult neural stem and progenitor cells and promotes proliferation through increased TrkB signaling, as we previously observed in neonatal neurogenesis (12).
Our study further reveals that loss of K v 1.1 in the neural stem cell lineage impedes adult-born granule cell development in later stages of adult neurogenesis. In K v 1.1 cKO mice, there was a reduction of DCX-expressing cells as well as impairment of adult-born granule cell maturation and positioning (Fig. 2  A-F ). Notably, aberrant positioning of adult-born granule cells has been observed in mouse models of traumatic brain injury, schizophrenia, and neurodegeneration (54)(55)(56). Failing to properly mature and position themselves, young adult-born granule cells produced from neural stem cells lacking K v 1.1 likely struggle to integrate into the dentate gyrus circuit, resulting in decreased survival of new adult-born granule cells and reduced mature adult-born granule cells (NeuN+) in K v 1.1 cKO mice (Fig. 2 G and H). Interestingly, a recent study discovered that postmortem samples from patients with neurodegenerative disease display a similar increase in radial glia-like neural stem cells and impairment in adult-born granule cell maturation and positioning (74). In mouse models, failures in adult neurogenesis often lead to deficits in hippocampus-dependent learning and memory (38,40,(60)(61)(62)(63)(64)(65). Indeed, K v 1.1 cKO mice have moderately diminished contextual fear learning and memory (Fig. 4). Together, these results underscore the critical function of K v 1.1 in maintaining adult-born granule cell maturation and positioning for proper integration into the dentate gyrus circuit and preservation of hippocampus-dependent learning and memory (Fig. 5B).
Our understanding of the role of K v 1.1 in adult neurogenesis also helps to clarify the role of K v 1.1 in neonatal neurogenesis. Given that we observed an initial increase in radial glia-like neural stem cell proliferation when K v 1.1 was removed from adult neural stem cells, the increase in neonatal neurogenesis observed when K v 1.1 is removed from neonatal neural stem cells in both K v 1.1 null mice (12) and K v 1.1 cKO mice (SI Appendix, Fig. S3) is likely to have arisen from neonatal radial glia-like neural stem cell overproliferation. Unlike adult neural stem and progenitor cells, neonatal neural stem and progenitor cells have extensive proliferative potential (43,44) and produce neurons with delayed cell death (57)(58)(59). These properties of neonatal neurogenesis allow the neuronal progenies to last for a longer period, which would account for the initial increase in K v 1.1 null progeny of Together, these factors contribute to the transient increase of K v 1.1 null progeny neurons in 2-to 3-mo-old but not 6-to 13-mo-old Nestin-Cre;Kcna1 +/À ;MADM-6 mice (Fig. 3). These results support our model that K v 1.1 functions to maintain hippocampal neurogenesis at multiple developmental timepoints.
In summary, we demonstrate that K v 1.1 is important for adult hippocampal neurogenesis and hippocampus-dependent contextual learning and memory. K v 1.1 likely regulates the neurogenic niche by preventing the overproliferation and depletion of radial glia-like neural stem cells. As young adult-born granule cells develop, loss of K v 1.1 impedes their dendritic maturation and positioning, likely hampering their integration into the circuit. These developmental failures in K v 1.1 cKO mice contribute to decreased adult-born granule cells and mild deficits in contextual fear learning and memory (Fig. 5). Our findings provide the basis for future studies to elucidate the impact of K v 1.1 regulation on adult neurogenesis under normal and pathophysiological conditions.

Materials and Methods
Animals. All experiments were approved by the Institutional Animal Care and Use Committees of the University of California, San Francisco and Academia Sinica. Two to five mice per cage were maintained in a temperature-controlled environment on a 12-h light/dark cycle with ad libitum access to food and water.
Kcna1 fl/fl mice (41) were obtained from E.G.'s laboratory at Southern Methodist University, Dallas, TX. Nestin-Cre ERT2 mice (38)(39)(40) were obtained from Mazen Kheirbek's laboratory at University of California, San Francisco, CA. PC::G5-tdT mice (42) were obtained from the Jackson Laboratory. We utilized the PC::G5-tdT reporter line because its Cre reporter alleles are located on a different chromosome (Chr 11) from Kcna1 (Chr 6). These three lines were bred together to create K v 1.1 cKO mice and maintained on a C57BL/6 background.
Drug Administration. For neonatal tamoxifen (MilliporeSigma) administration, tamoxifen was dissolved in 100% corn oil (MilliporeSigma) at 10 mg of tamoxifen per mL. At P0, pups were injected once subcutaneously with 30 μL of 10 mg of tamoxifen per mL. For adult tamoxifen administration, tamoxifen was dissolved in a solution of 10% ethanol (200 proof, VWR) in corn oil at 20 mg of tamoxifen per mL. Adult 8-wk-old mice were intraperitoneally injected with 100 mg of tamoxifen per kg of body weight once per day for three consecutive days for single-cell suspension and immunohistochemistry. Adult ∼4-mo-old mice were injected with 100 mg of tamoxifen per kg of body weight once per day for five consecutive days for behavioral experiments. BrdU (MilliporeSigma) was dissolved in sterile normal (0.9%) saline at 5 mg of BrdU per mL. One dose of 50 mg of BrdU per kg of body weight was injected subcutaneously at P7.
Single-Cell Suspension for qPCR and Electrophysiology. At 2 wk post tamoxifen injection, mice were euthanized, and their brains were transferred into ice-cold 1× Hanks' balanced salt solution (HBSS). Under a dissecting microscope, dentate gyri were isolated and pooled from four mice of the same genotype. The Neural Tissue Dissociation (P) Kit (Miltenyi Biotec) was used to dissociate tissue into single cells. After a final HBSS wash, the cell pellet was resuspended in 800 μL of Hibernate A Low Fluorescence medium (BrainBits). Detailed methods for qPCR and electrophysiology are included in SI Appendix, SI Materials and Methods.
Immunostaining. Mice were anesthetized with isoflurane (Henry Schein Animal Health) before transcardial perfusion with cold phosphate-buffered saline (PBS) followed by cold 4% paraformaldehyde (PFA) in PBS. Brains were removed, postfixed overnight in 4% PFA for ∼24 h at 4°C, washed in PBS, and immersed in 30% sucrose in PBS for a minimum of 48 h at 4°C for cryoprotection. Then, the brains were frozen in optimal cutting temperature compound (Fisher Scientific). Free-floating 30-μm sagittal sections were collected from the lineage tracing cohort and coronal sections were collected from the behavior cohort into PBS using a cryostat (Lecia CM3050 S, Leica Microsystems). Afterward, the sections were transferred into cryoprotectant (30% ethylene glycol, 30% glycerol, 40% PBS) and stored at À20°C.
Microscopy and Sampling. One of every 10 sections in a series spanning the entire dentate gyrus was imaged on a confocal microscope (Leica Sp8) using a 40× or 63× Harmonic Compound Plan Apochromat oil Confocal Scanning 2 objective. For lineage analysis, a z-stack of five 3-μm steps was collected per dentate gyrus section for one hemisphere. The dorsal dentate gyrus was quantified, given that Cre expression was variable in the ventral dentate gyrus (77). The upper third of the dentate granule cell layer was defined as being within two dentate granule cell layers of the molecular layer. For behavioral analysis, two sections from both hemispheres were quantified. Images were processed and analyzed using Fiji (ImageJ, NIH). Experimenters were blinded to the genotype of the mice during analysis.
Behavioral Tests. We did not observe overt differences in health between the two genotypes. Mice used for these experiments were healthy, without injuries that would interfere with behavioral testing. Behavioral data were obtained with the help of the Gladstone Behavioral Core. The experimenters were blinded to the genotype of the mice for all behavioral testing.
For behavior experiments, a cohort of adult ∼4-mo-old (age range 13 wk to 17 wk) mice were injected with tamoxifen once per day for five consecutive days. Five weeks after the last tamoxifen injection, behavioral testing began with the elevated plus maze, followed by the open field. Contextual conditioning and discrimination testing began at ∼6.5 wk post tamoxifen injection. Afterward, at ∼10.5 wk post tamoxifen injection, the hot plate test was conducted. Finally, mice were perfused at ∼17.5 wk post tamoxifen injection. Detailed methods for elevated plus maze, open field, contextual fear conditioning and discrimination, and hot plate behavioral tests are included in SI Appendix, SI Materials and Methods.
Statistical Analyses. All data were summarized as mean ± SEM. Comparisons between two genotypes were analyzed by unpaired two-tailed Student's t tests. If the data did not meet the Student's t test's assumptions of normality and variance, the data were analyzed using the unpaired two-tailed Student's t test with Welch's correction or the nonparametric Mann-Whitney U test, as indicated in the figure legends. Multiple group comparisons were assessed using one-way ANOVA with Holm-Sidak correction for multiple comparisons, two-way ANOVA followed by Sidak's multiple comparisons test, or linear mixed-model with restricted maximum likelihood (REML) and Geisser-Greenhouse correction followed by Sidak's multiple comparisons test, as indicated in the figure legends. The null hypothesis was rejected at P > 0.05. Data were analyzed using Prism 9 (GraphPad).
Data Availability. All study data are included in the article and/or SI Appendix.